Biological sex impacts oxidative stress in skeletal muscle in a porcine heat stress model.

Oxidative stress contributes to heat stress (HS)-mediated alterations in skeletal muscle; however, the extent to which biological sex mediates oxidative stress during HS remains unknown. We hypothesized muscle from males would be more resistant to oxidative stress caused by HS than muscle from females. To address this, male and female pigs were housed in thermoneutral conditions (TN; 20.8 ± 1.6°C; 62.0 ± 4.7% relative humidity; n = 8/sex) or subjected to HS (39.4 ± 0.6°C; 33.7 ± 6.3% relative humidity) for 1 (HS1; n = 8/sex) or 7 days (HS7; n = 8/sex) followed by collection of the oxidative portion of the semitendinosus. Although HS increased muscle temperature, by 7 days, muscle from heat-stressed females was cooler than muscle from heat-stressed males (0.3°C; P < 0.05). Relative protein abundance of 4-hydroxynonenal (4-HNE)-modified proteins increased in HS1 females compared with TN (P = 0.05). Furthermore, malondialdehyde (MDA)-modified proteins and 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentration, a DNA damage marker, was increased in HS7 females compared with TN females (P = 0.05). Enzymatic activities of catalase and superoxide dismutase (SOD) remained similar between groups; however, glutathione peroxidase (GPX) activity decreased in HS7 females compared with TN and HS1 females (P ≤ 0.03) and HS7 males (P = 0.02). Notably, HS increased skeletal muscle Ca2+ deposition (P = 0.05) and was greater in HS1 females compared with TN females (P < 0.05). Heat stress increased sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)2a protein abundance (P < 0.01); however, Ca2+ ATPase activity remained similar between groups. Overall, despite having lower muscle temperature, muscle from heat-stressed females had increased markers of oxidative stress and calcium deposition than muscle from males following identical environmental exposure.NEW & NOTEWORTHY Heat stress is a global threat to human health and agricultural production. We demonstrated that following 7 days of heat stress, skeletal muscle from females was more susceptible to oxidative stress than muscle from males in a porcine model, despite cooler muscle temperatures. The vulnerability to heat stress-induced oxidative stress in females may be driven, at least in part, by decreased antioxidant capacity and calcium dysregulation.

The effect of Mitoquinol (MitoQ) on heat stressed skeletal muscle from pigs, and a potential confounding effect of biological sex.

Heat stress (HS) poses a major threat to human health and agricultural production. Oxidative stress and mitochondrial dysfunction appear to play key roles in muscle injury caused by HS. We hypothesized that mitoquinol (MitoQ), would alleviate oxidative stress and cellular dysfunction in skeletal muscle during HS. To address this, crossbred barrows (male pigs) were treated with placebo or MitoQ (40 mg/d) and were then exposed to thermoneutral (TN; 20 °C) or HS (35 °C) conditions for 24 h. Pigs were euthanized following the environmental challenge and the red portion of the semitendinosus (STR) was collected for analysis. Unexpectedly, malondialdehyde concentration, an oxidative stress marker, was similar between environmental and supplement treatments. Heat stress decreased LC3A/B-I (p < 0.05) and increased the ratio of LC3A/B-II/I (p < 0.05), while p62 was similar among groups suggesting increased degradation of autophagosomes during HS. These outcomes were in disagreement with our previous results in muscle from gilts (female pigs). To probe the impact of biological sex on HS-mediated injury in skeletal muscle, we compared STR from these barrows to archived STR from gilts subjected to a similar environmental intervention. We confirmed our previous findings of HS-mediated dysfunction in muscle from gilts but not barrows. These data also raise the possibility that muscle from gilts is more susceptible to environment-induced hyperthermia than muscle from barrows.

Dietary Calcium and Phosphorus Amounts Affect Development and Tissue-Specific Stem Cell Characteristics in Neonatal Pigs.

BACKGROUND: Dietary calcium and phosphorus are required for bone and muscle development. Deficiencies of these macrominerals reduce bone mineral and muscle accretion potentially via alterations of mesenchymal stem cell (MSC) and satellite cell (SC) activities. OBJECTIVES: With increasing interest in the role of early-life events on lifetime health outcomes, we aimed to elucidate the impact of dietary calcium and phosphorus, from deficiency through excess, on MSC and SC characteristics during neonatal development. METHODS: Neonatal pigs [30 females, 1-d-old, 1.46 ± 0.04 kg body weight (BW)] were fed milk replacers for 16 d that were isonitrogenous and isocaloric with a consistent ratio of calcium to phosphorus, but either 25% deficient (calcium: 0.78%; phosphorus: 0.60%; CaPD), adequate (calcium: 1.08%; phosphorus: 0.84%; CaPA), or 25% in excess (calcium: 1.38%; phosphorus: 1.08%; CaPE) of calcium and phosphorus requirements based on sow-milk composition and extrapolation from NRC requirements for older pigs. BW and feed intake were recorded daily. Blood was collected for serum phosphorus, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23) determination. Humeri were collected for MSC isolation and radii/ulnae bone were collected for analysis. Longissimus dorsi muscle was collected for SC isolation and analysis. RESULTS: There was 4.6% increase in bone ash percentage in CaPE- versus CaPD-fed pigs (P < 0.05). In vivo proliferation indicated a 41.3% increase in MSCs in CaPA compared with CaPD and a 19% increase in SCs in CaPA compared with both CaPE and CaPD. MSCs from CaPD had 2- to 5-fold greater expression of peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), and lipoprotein lipase (LPL) but lower osteocalcin (BGLAP) and fibronectin (FN1) expression than CaPA (P < 0.05). SCs from CaPD-fed pigs had 19% lower in vivo proliferation than in CaPA-fed pigs. CONCLUSIONS: These findings demonstrated that feeding a diet marginally deficient in calcium and phosphorus to neonatal pigs had a great impact on bone development, MSC, and SC characteristics. These dietary deficiencies may program future bone health and muscle development by altering MSC and SC activities.

Lipid Intake Enhances Muscle Growth But Does Not Influence Glucose Kinetics in 3-Week-Old Low-Birth-Weight Neonatal Pigs.

BACKGROUND: Low-birth-weight (LBWT) neonates grow at a slower rate than their normal-birth-weight (NBWT) counterparts and may develop hypoglycemia postnatally. OBJECTIVE: We investigated whether dietary lipid supplementation would enhance growth and improve glucose production in LBWT neonatal pigs. METHODS: Twelve 3-d-old NBWT (1.606 kg) crossbred pigs were matched to 12 LBWT (1.260 kg) same-sex littermates. At 6 d of age, 6 pigs in each group were fed a low-energy (LE) or a high-energy (HE) isonitrogenous formula containing 5.2% and 7.3% fat, respectively. Body composition was assessed using dual-energy X-ray absorptiometry; plasma glucose and glycerol kinetics were assessed using stable isotope tracers. After killing, weights of skeletal muscles and visceral organs were measured. Data were analyzed by ANOVA for a 2 × 2 factorial design; temporal effects were investigated using repeated-measures analysis. RESULTS: Lipid supplementation did not affect body weight of LBWT or NBWT pigs. However, liver and longissimus dorsi weights as a percentage of body weight were greater for pigs fed an HE diet than for those fed an LE diet (4.3% compared with 3.4% and 1.5% compared with 1.2%, respectively) but remained less for LBWT than for NBWT pigs (3.8% compared with 3.9% and 1.3% compared with 1.5%, respectively) (P < 0.05). In addition, hepatic fat content increased (7.9 compared with 2.6 g) in pigs fed the HE compared with those fed the LE formula (P < 0.05). Lipid supplementation did not influence plasma glucose concentration which remained lower in the LBWT than in the NBWT group (4.1 compared with 4.5 mmol/L) (P < 0.05). CONCLUSIONS: Our data suggest that lipid supplementation modestly improved growth of skeletal muscle and the liver but did not affect glucose homeostasis in all groups, and glucose concentration remained lower in LBWT than in NBWT pigs. These data suggest that the previously reported hyperglycemic effect of lipid supplementation may depend on the route of administration or age of the neonatal pig.

Heat stress decreases metabolic flexibility in skeletal muscle of growing pigs.

Heat-stressed pigs experience metabolic alterations, including altered insulin profiles, reduced lipid mobilization, and compromised intestinal integrity. This is bioenergetically distinct from thermal neutral pigs on a similar nutritional plane. To delineate differences in substrate preferences between direct and indirect (via reduced feed intake) heat stress effects, skeletal muscle fuel metabolism was assessed. Pigs (35.3 ± 0.8 kg) were randomly assigned to three treatments: thermal neutral fed ad libitum (TN; 21°C, n = 8), heat stress fed ad libitum (HS; 35°C, n = 8), and TN, pair-fed/HS intake (PF; n = 8) for 7 days. Body temperature (TB) and feed intake (FI) were recorded daily. Longissimus dorsi muscle was biopsied for metabolic assays on days -2, 3, and 7 relative to initiation of environmental treatments. Heat stress increased TB and decreased FI ( P < 0.05). Heat stress inhibited incomplete fatty acid oxidation and glucose oxidation ( P < 0.05). Metabolic flexibility decreased in HS pigs compared with TN and PF controls ( P < 0.05). Both phosphofructokinase and pyruvate dehydrogenase (PDH) activities increased in PF ( P < 0.05); however, TN and HS did not differ. Heat stress inhibited citrate synthase and ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activities ( P < 0.05). Heat stress did not alter PDH phosphorylation or carnitine palmitoyltransferase 1 abundance but reduced acetyl-CoA carboxylase 1 (ACC1) protein abundance ( P < 0.05). In conclusion, HS decreased skeletal muscle fatty acid oxidation and metabolic flexibility, likely involving ß-HAD and ACC regulation.

Short-term heat stress results in increased apoptotic signaling and autophagy in oxidative skeletal muscle in Sus scrofa.

Prolonged environment-induced hyperthermia causes morbidities and mortality in humans and animals and appears to cause organ-specific injury and dysfunction. We have previously determined autophagic dysfunction and apoptotic signaling in oxidative skeletal muscle following prolonged hyperthermia. The aim of this investigation was to extend our knowledge regarding the early chronology of heat stress-mediated apoptotic and autophagic signaling in oxidative skeletal muscle. We hypothesized that 2, 4, and 6 h of hyperthermia would increase apoptosis and autophagy in oxidative skeletal muscle compared to thermoneutral (TN) conditions. Pigs were assigned to four groups (n = 8/group) and exposed to environmental heat stress (37 °C) for 0, 2, 4, or 6 h. Immediately following environmental exposure animals were euthanized and the red portion of the semitendinosus was collected. Markers of apoptotic signaling were increased following 2 h of heating but returned to baseline thereafter, while caspase 3 activity remained elevated 2-3 fold (p < .05) throughout the hyperthermic period. Heat stress increased (p < .05) markers of autophagic activation, and nucleation as well as autophagosome formation and degradation linearly throughout the heating intervention. In addition, 6 h of hyperthermia increased (p < .05) markers of mitophagy. These data suggest that apoptotic signaling precedes increased autophagy during acute heat stress in oxidative skeletal muscle.

Prolonged environment-induced hyperthermia alters autophagy in oxidative skeletal muscle in Sus scrofa.

Prolonged heat stress represents a continuing threat to human health and agricultural production. Despite the broad, negative impact of prolonged hyperthermia little is known about underlying pathological mechanisms leading to negative health outcomes, which has limited the development of etiological interventions and left clinicians and producers with only cooling and rehydration strategies. The purpose of this investigation was to determine the extent to which prolonged environment-induced hyperthermia altered autophagy in oxidative skeletal muscle in a large animal model, serving the dual purpose of accurately modeling human physiology as well as agricultural production. We hypothesized that prolonged hyperthermia would induce autophagy in skeletal muscle, independent of the accompanying caloric restriction. To test this hypothesis pigs were treated as follows: thermoneutral (20 °C), heat stress (35 °C), or were held under thermoneutral conditions but pair-fed to the heat stress group for seven days. Upon euthanasia the red portion of the semitendinosus was collected. We found that prolonged hyperthermic exposure increased oxidative stress without a corresponding change in antioxidant enzyme activities. Hyperthermia prevented initiation of autophagy despite increased markers of nucleation, elongation and autophagosome formation. However, p62 relative protein abundance, which is inversely correlated with autophagic degradation, was strongly increased suggesting suppressed degradation of autophagosomes. Markers of mitophagy and mitochondrial abundance were largely similar between groups. These data indicate that faulty autophagy plays a key role in hyperthermic muscle dysfunction.

Twelve hours of heat stress induces inflammatory signaling in porcine skeletal muscle.

Heat stress causes morbidity and mortality in humans and animals and threatens food security by limiting livestock productivity. Inflammatory signaling may contribute to heat stress-mediated skeletal muscle dysfunction. Previously, we discovered increased circulating endotoxin and intramuscular oxidative stress and TNF-α protein abundance, but not inflammatory signaling following 24 and 72 h of heat stress. Thus the purpose of this investigation was to clarify the role of inflammatory signaling in heat-stressed skeletal muscle. Crossbred gilts (n = 8/group) were assigned to either thermal neutral (24°C), heat stress (37°C), or pair-fed thermal neutral (24°C) conditions for 12 h. Following treatment, animals were euthanized, and the semitendinosus red (STR) and white (STW) were recovered. Heat stress did not alter inflammatory signaling in STW. In STR, relative heat shock protein abundance was similar between groups, as was nuclear content of heat shock factor 1. In whole homogenate, relative abundance of the NF-κB activator inhibitory κB kinase-α was increased by heat stress, although abundance of NF-κB was similar between groups. Relative abundance of phosphorylated NF-κB was increased by heat stress in nuclear fractions. Activator protein-1 (AP-1) signaling was similar between groups. While there were few differences in transcript expression between thermal neutral and heat stress, 80 and 56% of measured transcripts driven by NF-κB or AP-1, respectively, were increased by heat stress compared with pair-fed thermal neutral. Heat stress also caused a reduction in IL-6 transcript and relative protein abundance. These data demonstrate that short-term heat stress causes inflammatory signaling through NF-κB in oxidative, but not glycolytic, skeletal muscle.

Heat Stress Alters Ovarian Insulin-Mediated Phosphatidylinositol-3 Kinase and Steroidogenic Signaling in Gilt Ovaries.

Heat stress (HS) compromises a variety of reproductive functions in several mammalian species. Inexplicably, HS animals are frequently hyperinsulinemic despite marked hyperthermia-induced hypophagia. Our objectives were to determine the effects of HS on insulin signaling and components essential to steroid biosynthesis in the pig ovary. Female pigs (35 ± 4 kg) were exposed to constant thermoneutral (20°C; 35%-50% humidity; n = 6) or HS conditions (35°C; 20%-35% humidity; n = 6) for either 7 (n = 10) or 35 days (n = 12). After 7 days, HS increased (P < 0.05) ovarian mRNA abundance of the insulin receptor (INSR), insulin receptor substrate 1 (IRS1), protein kinase B subunit 1 (AKT1), low-density lipoprotein receptor (LDLR), luteinizing hormone receptor (LHCGR), and aromatase (CYP19a). After 35 days, HS increased INSR, IRS1, AKT1, LDLR, LHCGR, CYP19a, and steroidogenic acute regulatory protein (STAR) ovarian mRNA abundance. In addition, after 35 days, HS increased ovarian phosphorylated IRS1 (pIRS1), phosphorylated AKT (pAKT), STAR, and CYP19a protein abundance. Immunostaining analysis revealed similar localization of INSR and pAKT1 in the cytoplasmic membrane and oocyte cytoplasm, respectively, of all stage follicles, and in theca and granulosa cells. Collectively, these results demonstrate that HS alters ovarian insulin-mediated PI3K signaling pathway members, which likely impacts follicle activation and viability. In summary, environmentally induced HS is an endocrine-disrupting exposure that modifies ovarian physiology and potentially compromises production of ovarian hormones essential for fertility and pregnancy maintenance.

The impact of in utero heat stress and nutrient restriction on progeny body composition.

We recently demonstrated that in utero heat stress (IUHS) alters future tissue accretion in pigs, but whether this is a conserved response among species, is due to the direct effects of heat stress (HS) or mediated by reduced maternal feed intake (FI) is not clear. Study objectives were to compare the quantity and rate of tissue accretion in rats exposed to differing in utero thermal environments while eliminating the confounding effect of dissimilar maternal FI. On d3 of gestation, pregnant Sprague-Dawley rats (189.0±5.9g BW) were exposed to thermoneutral (TN; 22.2±0.1°C; n=8), or HS conditions (cyclical 30 to 34°C; n=8) until d18 of gestation. A third group was pair-fed to HS dams in TN conditions (PFTN; 22.2±0.1°C; n=8) from d4 to d19 of gestation. HS increased dam rectal temperature (p=0.01; 1.3°C) compared to TN and PFTN mothers, and reduced FI (p=0.01; 33%) compared to TN ad libitum fed controls. Although litter size was similar (p=0.97; 10.9 pups/litter), pup birth weight was reduced (p=0.03; 15.4%) in HS compared to PFTN and TN dams. Two male pups per dam [n=8 in utero TN (IUTN); n=8 IUHS; n=8 in utero PFTN (IUPFTN)] were selected from four dams per treatment based on similar gestation length, and body composition was determined using dual-energy x-ray absorptiometry (DXA) on d26, d46, and d66 of postnatal life. Whole-body fat content increased (p=0.01; 11.2%), and whole-body lean tissue decreased (p=0.01; 2.6%) in IUPFTN versus IUTN and IUHS offspring. Whole-body composition was similar between IUHS and IUTN offspring. Epididymal fat pad weight increased (p=0.03; 21.6%) in IUPFTN versus IUHS offspring. In summary and in contrast to pigs, IUHS did not impact rodent body composition during this stage of growth; however, IUPFTN altered the future hierarchy of tissue accretion.

Myoblasts from intrauterine growth-restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres.

Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P < 0.05) than controls and had smaller (P < 0.05) carcasses and semitendinosus myofibre areas. IUGR semitendinosus muscles had similar percentages of pax7-positive nuclei and pax7 mRNA but lower (P < 0.05) percentages of myogenin-positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer (P < 0.05) proliferating myoblasts (PNCA-positive-pax7-positive) than controls (44 ± 2% vs. 52 ± 1%). Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer (P < 0.05) myoD-positive cells than controls and replicated 20% less (P < 0.05) during a 2 h BrdU pulse. IUGR myoblasts also replicated less (P < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P < 0.05) with IUGR FSS-supplemented media compared to control FSS-supplemented media. In differentiation-promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin-positive nuclei after 5 days and formed similar-sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofibre size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR newborn offspring.

Heat stress alters hematological parameters in barrows and gilts.

The purpose of this investigation was to establish the role biological sex plays in circulating factors following heat stress (HS). Barrows and gilts (36.8 ±â€…3.7 kg body weight) were kept in either thermoneutral (TN; 20.8 ±â€…1.6 °C; 62.0% ±â€…4.7% relative humidity; n = 8/sex) conditions or exposed to HS (39.4 ±â€…0.6 °C; 33.7% ±â€…6.3% relative humidity) for either 1 (HS1; n = 8/sex) or 7 (HS7; n = 8/sex) d. Circulating glucose decreased as a main effect of the environment (P = 0.03). Circulating non-esterified fatty acid (NEFA) had an environment × sex interaction (P < 0.01) as HS1 barrows had increased NEFA compared to HS1 gilts (P = 0.01) and NEFA from HS7 gilts increased compared to HS1 gilts (P = 0.02) and HS7 barrows (P = 0.04). Cortisol, insulin, glucagon, T3, and T4 were reduced as a main effect of environment (P ≤ 0.01). Creatinine was increased in HS1 and HS7 animals compared to TN (P ≤ 0.01), indicative of decreased glomerular filtration rate. White blood cell populations exhibited differential patterns based on sex and time. Neutrophils and lymphocytes had an environment × sex interaction (P ≤ 0.05) as circulating neutrophils were increased in HS1 barrows compared to TN and HS7 barrows, and HS1 gilts (P ≤ 0.01) and HS7 barrows had less neutrophils compared to TN barrows (P = 0.01), whereas they remained similar in gilts. In contrast, barrow lymphocyte numbers were similar between groups, but in HS7 gilts they were decreased compared to TN and HS1 gilts (P ≤ 0.04). In total, these data demonstrate that HS alters a host of circulating factors and that biological sex mediates, at least in part, the physiological response to HS.

Heat stress (HS) negatively impacts efficient pork production; however, the role of biological sex is largely unknown. The objective of this study was to determine the extent to which HS differentially impacted hematological parameters in barrows and gilts. To address this, 3-mo-old barrows and gilts were exposed to ambient temperature (TN) or constant HS for 1 or 7 d. Following the experimental period, blood was collected for analysis of hormones, metabolites, immune cells, and markers of organ damage. Overall, cortisol, insulin, glucagon, T3, and T4 were reduced following HS. Furthermore, 7 d of HS decreased circulating glucose, albeit slightly. Circulating fatty acids had a sex-specific response as HS1 barrows and HS7 gilts were increased compared to their environmental counterparts, though, these changes are minor compared to those expected with a similar feed restriction. HS caused immune system activation in barrows and gilts; however, circulating levels of specific white blood cells were time- and sex-dependent. Barrows appeared more resistant to HS-mediated kidney injury acutely; however, with continued heating, markers of kidney injury were similar between barrows and gilts. In total, these data suggest biological sex regulates some, but not all, aspects of HS-mediated biological changes in pigs.

Therapeutic effects of mitoquinol during an acute heat stress challenge in growing barrows.

Study objectives were to determine the effects of mitoquinol (MitoQ, a mitochondrial-targeted antioxidant) on biomarkers of metabolism and inflammation during acute heat stress (HS). Crossbred barrows [n = 32; 59.0 ±â€…5.6 kg body weight (BW)] were blocked by BW and randomly assigned to 1 of 4 environmental-therapeutic treatments: 1) thermoneutral (TN) control (n = 8; TNCon), 2) TN and MitoQ (n = 8; TNMitoQ), 3) HS control (n = 8; HSCon), or 4) HS and MitoQ (n = 8; HSMitoQ). Pigs were acclimated for 6 d to individual pens before study initiation. The trial consisted of two experimental periods (P). During P1 (2 d), pigs were fed ad libitum and housed in TN conditions (20.6 ±â€…0.8 °C). During P2 (24 h), HSCon and HSMitoQ pigs were exposed to continuous HS (35.2 ±â€…0.2 °C), while TNCon and TNMitoQ remained in TN conditions. MitoQ (40 mg/d) was orally administered twice daily (0700 and 1800 hours) during P1 and P2. Pigs exposed to HS had increased rectal temperature, skin temperature, and respiration rate (+1.5 °C, +6.8 °C, and +101 breaths per minute, respectively; P < 0.01) compared to their TN counterparts. Acute HS markedly decreased feed intake (FI; 67%; P < 0.01); however, FI tended to be increased in HSMitoQ relative to HSCon pigs (1.5 kg vs. 0.9 kg, respectively; P = 0.08). Heat-stressed pigs lost BW compared to their TN counterparts (-4.7 kg vs. +1.6 kg, respectively; P < 0.01); however, the reduction in BW was attenuated in HSMitoQ compared to HSCon pigs (-3.9 kg vs. -5.5 kg, respectively; P < 0.01). Total gastrointestinal tract weight (empty tissue and luminal contents) was decreased in HS pigs relative to their TN counterparts (6.2 kg vs. 8.6 kg, respectively; P < 0.01). Blood glucose increased in HSMitoQ relative to HSCon pigs (15%; P = 0.04). Circulating non-esterified fatty acids (NEFA) increased in HS compared to TN pigs (P < 0.01), although this difference was disproportionately influenced by elevated NEFA in HSCon relative to HSMitoQ pigs (251 µEq/L vs. 142 µEq/L; P < 0.01). Heat-stressed pigs had decreased circulating insulin relative to their TN counterparts (47%; P = 0.04); however, the insulin:FI ratio tended to increase in HS relative to TN pigs (P = 0.09). Overall, circulating leukocytes were similar across treatments (P > 0.10). Plasma C-reactive protein remained similar among treatments; however, haptoglobin increased in HS relative to TN pigs (48%; P = 0.03). In conclusion, acute HS exposure negatively altered animal performance, inflammation, and metabolism, which were partially ameliorated by MitoQ.

Heat stress (HS) compromises animal health and productivity, and this causes major economic losses in almost every livestock sector. The negative consequences of HS are thought to originate from intestinal barrier dysfunction and subsequent immune activation. The underlying causes of lost intestinal integrity during HS are likely multifactorial; however, intestinal ischemia, increased accumulation of reactive oxygen species, and the ensuing epithelial oxidative damage might be potential causes. Mitochondria-targeted antioxidants, such as mitoquinol (MitoQ), are probably more effective than traditional dietary antioxidants (i.e., selenium, vitamin E) at alleviating oxidative stress, as they localize and accumulate within the mitochondria, potentiating their antioxidant activity. Thus, the present study aimed to investigate MitoQ's role during a thermal event in growing pigs. Herein, HS increased all body temperature indices, decreased feed intake (FI), and induced substantial body weight (BW) loss. Interestingly, the reduction in FI and BW was less dramatic in pigs receiving MitoQ. Changes in circulating metabolism and the acute phase response were observed due to the HS challenge; however, contrary to our expectations, these changes were not offset by MitoQ administration. Although our results suggest a positive MitoQ effect on growth performance, future studies are needed to corroborate the replicability of this response during HS.

Skeletal muscle satellite cell migration to injured tissue measured with 111In-oxine and high-resolution SPECT imaging.

The delivery of adult skeletal muscle stem cells, called satellite cells, to several injured muscles via the circulation would be useful, however, an improved understanding of cell fate and biodistribution following their delivery is important for this goal to be achieved. The objective of this study was to evaluate the ability of systemically delivered satellite cells to home to injured skeletal muscle using single-photon emission computed tomography (SPECT) imaging of (111)In-labeled satellite cells. Satellite cells labeled with (111)In-oxine and green fluorescent protein (GFP) were injected intravenously after bupivicaine-induced injury to the tibialis anterior muscle. Animals were imaged with a high-resolution SPECT system called FastSPECT II for up to 7 days after transplantation. In vivo FastSPECT II imaging demonstrated a three to five-fold greater number of transplanted satellite cells in bupivicaine-injured muscle as compared to un-injured muscle after transplantation; a finding that was verified through autoradiograph analysis and quantification of GFP expression. Satellite cells also accumulated in other organs including the lung, liver, and spleen, as determined by biodistribution measurements. These data support the ability of satellite cells to home to injured muscle and support the use of SPECT and autoradiograph imaging techniques to track systemically transplanted (111)In labeled satellite cells in vivo, and suggest their homing may be improved by reducing their entrapment in filter organs.

Reproductive and lactational responses of multiparous dairy cattle to short-term postpartum chromium supplementation during the summer months.

The objective of this work was to evaluate the potential benefits of short-duration, high-dose chromium (Cr) supplementation in early postpartum dairy cows during the summer months. Multiparous, early-lactation cows (20.95 ± 0.21 d in milk) were assigned to 1 of 2 treatment groups: (1) control diet (Con; n = 10) or (2) control diet + Cr propionate (CrPro; 12 mg/head per day Cr; n = 12). Measurements of ovarian structures, respiration rates (RR), rectal temperatures (RT), and blood glucose concentrations were recorded every 3 d. Blood was also collected for analysis of plasma progesterone concentrations. Every 6 d, in conjunction with ultrasonography, endometrial cytology samples were collected via cytobrush from each cow to determine the incidences of subclinical endometritis, as determined by polymorphonuclear leukocyte (PMNL)%. No differences were detected in RR, RT, blood glucose, feed intake, milk yield, or change in body weight. The supplementation did, however, improve some reproductive parameters. At cytology sample 6, the PMNL% increased in Con cows, and was greater than the PMNL% in the CrPro group. Chromium consumption did not affect the number or size of most follicles, with the exception being the 6 to 9 mm category where the CrPro group had a greater average diameter and tended to have greater numbers of follicles in this category. While corpus luteum numbers and size did not differ between treatments, the ratio of progesterone to average corpus luteum volume was greater in the CrPro group compared with the Con group. The results from this study indicate that, whereas the short-term, high-dose supplementation strategy did not affect feed intake or milk yield, this Cr supplementation strategy could benefit reproductive performance during periods of stress.

The Probiotic Bacillus subtilis MB40 Improves Immunity in a Porcine Model of Listeriosis.

Probiotics for humans and direct-fed microbials for livestock are increasingly popular dietary ingredients for supporting immunity. The aim of this study was to determine the effects of dietary supplementation of Bacillus subtilis MB40 (MB40) on immunity in piglets challenged with the foodborne pathogen Listeria monocytogenes (LM). Three-week-old piglets (n = 32) were randomly assigned to four groups: (1) basal diet, (2) basal diet with LM challenge, (3) MB40-supplemented diet, and (4) MB40-supplemented diet with LM challenge. Experimental diets were provided throughout a 14-day (d) period. On d8, piglets in groups 2 and 4 were intraperitoneally inoculated with LM at 108 CFU/mL per piglet. Blood samples were collected at d1, d8, and d15 for biochemical and immune response profiling. Animals were euthanized and necropsied at d15 for liver and spleen bacterial counts and intestinal morphological analysis. At d15, LM challenge was associated with increased spleen weight (p = 0.017), greater circulating populations of neutrophils (p = 0.001) and monocytes (p = 0.008), and reduced ileal villus height to crypt depth ratio (p = 0.009), compared to non-challenged controls. MB40 supplementation reduced LM bacterial counts in the liver and spleen by 67% (p < 0.001) and 49% (p < 0.001), respectively, following the LM challenge, compared to the basal diet. MB40 supplementation was also associated with decreased circulating concentrations of monocytes (p = 0.007). Altogether, these data suggest that MB40 supplementation is a safe and well-tolerated approach to enhance immunity during systemic Listeria infection.

Short-term consumption of the mycotoxin zearalenone by pubertal gilts causes persistent changes in the histoarchitecture of reproductive tissues.

Consumption of zearalenone (ZEN) detrimentally affects tissues and systems throughout the body, and these deleterious effects are especially pronounced in swine. The objectives of this project were to determine the effects of short-term consumption of ZEN (at concentrations that could be found on-farm) on growth, carcass weight, liver weight, and reproductive tissues of pubertal gilts, and to determine if the effects are transient or persistent. Cross-bred gilts (107.25 ± 2.69 kg) were randomly assigned to one of three feed treatments: 1) solvent only for 21 d (CON; n = 10), 2) ZEN for 7 d followed by 14 d of solvent (ZEN-7; 6 mg/d; n = 10), and 3) ZEN for 21 d (ZEN-21; 6 mg/d; n = 10). Body weights were collected at the beginning and end of the experiment (189.1 ± 0.8 and 211.1 ± 0.8 d of age, respectively). Carcass weights and tissues were collected at harvest. There were no treatment-based differences in growth, carcass, liver, or reproductive tissue weights. Histological analyses revealed differences based on treatment and the interaction between treatment and luteal status. The thickness of the ampullary muscularis declined with ZEN exposure (P < 0.05), while the isthmic epithelial cell height (P < 0.01) and uterine endometrial thickness (P < 0.02) increased. Interestingly, the thickness of the isthmic muscularis, uterine myometrium, and epithelial cell height only differed in the presence of a corpus luteum. Uterine epithelial cell height in the luteal phase was lowest in ZEN-7 pigs (P < 0.01). The isthmic muscularis in the luteal phase was thinner in pigs from both ZEN treatments (P < 0.01). Conversely, the luteal-stage myometrium was thicker in pigs from both ZEN treatments (P < 0.01). The discovery of these tissue-based differences during the luteal phase is particularly concerning since this corresponds with the time when embryos would be affected by the functional competency of the oviduct and uterus. The results of this work demonstrate that short-term consumption of ZEN produces microscopic, but not macroscopic alterations in reproductive organs which are likely to have negative effects on their subsequent function and that these differences persist even after ZEN consumption ceases. Taken together, these results indicate that it is insufficient to rely solely on outwardly visible symptoms as indicators of zearalenone exposure, as detrimental effects on reproductive tissues were found in the absence of phenotypic and morphologic changes.

The mycotoxin zearalenone is a common contaminant of livestock feed. The consumption of zearalenone is particularly problematic for pigs as they are very sensitive to its effects. This study evaluated the effects of zearalenone on growth, carcass weight, liver weight, and reproductive tissues in young female pigs. Thirty pigs were split across three treatment groups. The control group was given standard feed (no zearalenone added) for 21 d, the second group received zearalenone-treated feed for 7 d followed by 14 d of standard feed, and the third group received zearalenone-treated feed for the full 21 d. Pigs receiving the treated feed exhibited no visible symptoms associated with zearalenone consumption. There were also no treatment-related differences in growth, carcass weight, liver weight, or reproductive tract weight. Histological analyses of both the oviduct and uterus revealed changes in tissue thickness that could indicate potential impairments in reproductive organ function. Changes in tissue layer thickness were especially prominent in the luteal phase. This interaction between the treatment and the presence of a corpus luteum is noteworthy because tract function during the luteal phase is imperative for fertilization and early embryonic development.

Supplementation with artificial sweetener and capsaicin alters metabolic flexibility and performance in heat-stressed and feed-restricted pigs.

Substantial economic losses in animal agriculture result from animals experiencing heat stress (HS). Pigs are especially susceptible to HS, resulting in reductions in growth, altered body composition, and compromised substrate metabolism. In this study, an artificial high-intensity sweetener and capsaicin (CAPS-SUC; Pancosma, Switzerland) were supplemented in combination to mitigate the adverse effects of HS on pig performance. Forty cross-bred barrows (16.2 ± 6 kg) were assigned to one of five treatments: thermal neutral controls (TN) (22 ± 1.2 °C; 38%-73% relative humidity) with ad libitum feed, HS conditions with ad libitum feed with (HS+) or without (HS-) supplementation, and pair-fed to HS with (PF+) or without supplementation (PF-). Pigs in heat-stressed treatments were exposed to a cyclical environmental temperature of 12 h at 35 ± 1.2 °C with 27%-45% relative humidity and 12 h at 30 ± 1.1 °C with 24%-35% relative humidity for 21 d. Supplementation (0.1 g/kg feed) began 7 d before and persisted through the duration of environmental or dietary treatments (HS/PF), which lasted for 21 d. Rectal temperatures and respiration rates (RR; breaths/minute) were recorded thrice daily, and feed intake (FI) was recorded daily. Before the start and at the termination of environmental treatments (HS/PF), a muscle biopsy of the longissimus dorsi was taken for metabolic analyses. Blood samples were collected weekly, and animals were weighed every 3 d during treatment. Core temperature (TN 39.2 ± 0.02 °C, HS- 39.6 ± 0.02 °C, and HS+ 39.6 ± 0.02 °C, P < 0.001) and RR (P < 0.001) were increased in both HS- and HS+ groups, but no difference was detected between HS- and HS+. PF- pigs exhibited reduced core temperature (39.1 ± 0.02 °C, P < 0.001), which was restored in PF+ pigs (39.3 ± 0.02 °C) to match TN. Weight gain and feed efficiency were reduced in PF- pigs (P < 0.05) but not in the PF+ or the HS- or HS+ groups. Metabolic flexibility was decreased in the HS- group (-48.4%, P < 0.05) but maintained in the HS+ group. CAPS-SUC did not influence core temperature or weight gain in HS pigs but did restore core temperature, weight gain, and feed efficiency in supplemented PF pigs. In addition, supplementation restored metabolic flexibility during HS and improved weight gain and feed efficiency during PF, highlighting CAPS-SUC's therapeutic metabolic effects.

Heat stress reduces pig performance due to metabolic responses to heat. During heat stress, pigs lose the ability to metabolize fatty acids for energy and rely on carbohydrates to fuel growth. Evidence has shown that capsaicin, the active ingredient in chili peppers, interacts with heat-sensing receptors to protect against heat stress by preventing changes to metabolism. Artificial sweeteners can also preserve fat metabolism by inducing the secretion of metabolic regulatory hormones from the gut. This study examined a combination of capsaicin and artificial sweetener to restore growth and maintain metabolism during 3 wk of heat stress. As pigs often reduce their feed intake during heat stress, a group of pigs was feed restricted to match the reduced feeding observed in the heat-stressed pigs. Pigs given the feed supplement during heat stress maintained their metabolic flexibility, a measure of metabolic health. In agreement with previous short-term studies, the capsaicin and artificial sweetener supplement improved feed efficiency and weight gain in feed-restricted pigs. This study demonstrated that supplementation with capsaicin and artificial sweetener may prevent metabolic dysfunction during heat stress. This study also confirmed that supplementation with capsaicin and artificial sweetener does improve feed-restricted pigs' growth and feed efficiency.

Effects of transforming growth factor-beta (TGF-ß1) on satellite cell activation and survival during oxidative stress.

The regulation of adult skeletal muscle repair and regeneration is largely due to the contribution of resident adult myogenic precursor cells called satellite cells. The events preceding their participation in muscle repair include activation (exit from quiescence), proliferation, and differentiation. This study examined the effects of transforming growth factor-beta (TGF-ß1) on satellite cell activation, determined whether TGF-ß1 could maintain quiescence in the presence of hepatocyte growth factor (HGF), and whether the regulation of satellite cell activation with TGF-ß1 improves the ability of satellite cells to withstand oxidative stress. The addition of TGF-ß1 during early satellite cell activation (0-48 h) or during the proliferative phase (48-96 h) maintained and induced satellite cell quiescence, respectively, as determined by myogenic differentiation (MyoD) protein expression. TGF-ß1 also attenuated satellite cell activation when used with HGF. Finally, the role of quiescence in protecting cells against oxidative stress was examined. TGF-ß1 treatment and the low pH satellite cell preparation procedure, a technique that forestalls spontaneous activation in vitro, both enhanced survival of cultured satellite cells following hydrogen peroxide treatment. These findings indicate that TGF-ß1 is capable of maintaining and inducing satellite cell quiescence and suggest methods to maintain satellite cell quiescence may improve their transplantation efficiency.

Rapamycin administration during an acute heat stress challenge in growing pigs.

Study objectives were to determine the effects of rapamycin (Rapa) on biomarkers of metabolism and inflammation during acute heat stress (HS) in growing pigs. Crossbred barrows (n = 32; 63.5 ± 7.2 kg body weight [BW]) were blocked by initial BW and randomly assigned to 1 of 4 environmental-therapeutic treatments: 1) thermoneutral (TN) control (n = 8; TNCon), 2) TN and Rapa (n = 8; TNRapa), 3) HS control (n = 8; HSCon), or 4) HS and Rapa (n = 8; HSRapa). Following 6 d of acclimation to individual pens, pigs were enrolled in two experimental periods (P). During P1 (10 d), pigs were fed ad libitum and housed in TN conditions (21.3 ± 0.2°C). During P2 (24 h), HSCon and HSRapa pigs were exposed to constant HS (35.5 ± 0.4°C), while TNCon and TNRapa pigs remained in TN conditions. Rapamycin (0.15 mg/kg BW) was orally administered twice daily (0700 and 1800 hours) during both P1 and P2. HS increased rectal temperature and respiration rate compared to TN treatments (1.3°C and 87 breaths/min, respectively; P < 0.01). Feed intake (FI) markedly decreased in HS relative to TN treatments (64%; P < 0.01). Additionally, pigs exposed to HS lost BW (4 kg; P < 0.01), while TN pigs gained BW (0.7 kg; P < 0.01). Despite marked changes in phenotypic parameters caused by HS, circulating glucose and blood urea nitrogen did not differ among treatments (P > 0.10). However, the insulin:FI increased in HS relative to TN treatments (P = 0.04). Plasma nonesterified fatty acids (NEFA) increased in HS relative to TN treatments; although this difference was driven by increased NEFA in HSCon compared to TN and HSRapa pigs (P < 0.01). Overall, circulating white blood cells, lymphocytes, and monocytes decreased in HS compared to TN pigs (19%, 23%, and 33%, respectively; P ≤ 0.05). However, circulating neutrophils were similar across treatments (P > 0.31). The neutrophil-to-lymphocyte ratio (NLR) was increased in HS relative to TN pigs (P = 0.02); however, a tendency for reduced NLR was observed in HSRapa compared to HSCon pigs (21%; P = 0.06). Plasma C-reactive protein tended to differ across treatments (P = 0.06) and was increased in HSRapa relative to HSCon pigs (46%; P = 0.03). Circulating haptoglobin was similar between groups. In summary, pigs exposed to HS had altered phenotypic, metabolic, and leukocyte responses; however, Rapa administration had limited impact on outcomes measured herein.